U.S. patent application number 12/383178 was filed with the patent office on 2010-09-23 for fluorescent saccharide conjugates.
Invention is credited to Donald L. Barbeau.
Application Number | 20100240878 12/383178 |
Document ID | / |
Family ID | 42738210 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100240878 |
Kind Code |
A1 |
Barbeau; Donald L. |
September 23, 2010 |
Fluorescent saccharide conjugates
Abstract
The present invention relates to improved fluorescently labeled
monosaccharide and low molecular weight oligosaccharide conjugates
for immunofluorescent staining, confocal microscopic imaging and
flow cytometry/fluorescence activated cell sorting (FACS). These
fluorophore conjugates target cells, components of cell surfaces
and extracellular components; and are useful as probes for tumor
targeting and cellular uptake.
Inventors: |
Barbeau; Donald L.;
(Evanston, IL) |
Correspondence
Address: |
Donald L. Barbeau
6 Williamsburg Circle
Evanston
IL
60203
US
|
Family ID: |
42738210 |
Appl. No.: |
12/383178 |
Filed: |
March 20, 2009 |
Current U.S.
Class: |
536/17.4 ;
536/123.1; 536/17.6 |
Current CPC
Class: |
C07H 15/26 20130101 |
Class at
Publication: |
536/17.4 ;
536/17.6; 536/123.1 |
International
Class: |
C07H 15/26 20060101
C07H015/26; C07H 1/00 20060101 C07H001/00 |
Claims
1. A compound having the formula ##STR00010## where R is an
alkylene or heteroalkylene, Y=0 or 1, R.sub.1, R.sub.2 and R.sub.3
are independently hydrogen, alkyl, aryl, aralkyl and cycloalkyl,
R.sub.4 is independently hydrogen, a monomeric glycoside, a dimeric
glycoside or alkyl, X=0 or 1, Z is oxygen or a linker, n is from 1
to about 4 and R.sub.5 is hydrogen or a fluorophore with the
proviso that said compound contains at least one fluorophore.
2. The compound of claim 1 having the formula ##STR00011##
3. The compound of claim 1 wherein said fluorophore is fluorescein
isothiocyanate, a rhodamine dye or Texas Red.
4. The compound of claim 2 wherein said fluorophore is fluorescein
isothiocyanate, a rhodamine dye or Texas Red.
5. The compound of claim 2 wherein R.sub.1, R.sub.2 and R.sub.3 are
hydrogen.
6. The compound of claim 2 wherein R.sub.1, R.sub.2 and R.sub.3 are
independently alkyl, aryl, aralkyl and cycloalkyl.
7. The compound of claim 2 having the formula ##STR00012##
8. The compound of claim 2 having the formula ##STR00013##
9. The compound of claim 1 having the formula ##STR00014##
10. A compound having the formula ##STR00015## where R is an
alkylene or heteroalkylene, Y=0 or 1, R.sub.1, R.sub.2 and R.sub.3
are independently hydrogen, alkyl, aryl, aralkyl and cycloalkyl,
X=0 or 1, Z is oxygen or a linker and R.sub.5 is a fluorophore.
11. The compound of claim 10 wherein said fluorophore is
fluorescein isothiocyanate, a rhodamine dye or Texas Red.
12. The compound of claim 10 wherein said fluorophore is
fluorescein isothiocyanate, a rhodamine dye or Texas Red.
13. The compound of claim 10 wherein R.sub.1, R.sub.2 and R.sub.3
are hydrogen.
14. The compound of claim 10 wherein R.sub.1, R.sub.2 and R.sub.3
are independently alkyl, aryl, aralkyl and cycloalkyl.
15. A compound having the formula ##STR00016## where R is an
alkylene or heteroalkylene, Y=0 or 1, R.sub.1, R.sub.2 and R.sub.3
are independently hydrogen, alkyl, aryl, aralkyl and cycloalkyl,
R.sub.4 is independently hydrogen, a monomeric glycoside, a dimeric
glycoside or alkyl, X=0 or 1, Z is oxygen or a linker, n is from 1
to about 4 and R.sub.5 is hydrogen or a fluorophore with the
proviso that said compound contains at least one fluorophore.
16. The compound of claim 15 having the formula ##STR00017##
17. The compound of claim 15 wherein said fluorophore is
fluorescein isothiocyanate, a rhodamine dye or Texas Red.
18. The compound of claim 15 wherein said fluorophore is
fluorescein isothiocyanate, a rhodamine dye or Texas Red.
19. The compound of claim 16 wherein R.sub.1, R.sub.2 and R.sub.3
are hydrogen.
20. The compound of claim 16 wherein R.sub.1, R.sub.2 and R.sub.3
are independently alkyl, aryl, aralkyl and cycloalkyl.
Description
BACKGROUND OF THE INVENTION
[0001] Fluorescent probes are routinely used to identify and
characterize populations of cells, cell surfaces and extracellular
components through the use of immunofluorescent staining,
fluorescence microscopy, confocal microscopic imaging and flow
cytometry/fluorescence activated cell sorting (FACS). In some
cases, fluorescent probes are directly conjugated to monoclonal
antibodies that can specifically bind to cell surface proteins,
lectins and carbohydrates. In other cases, fluorescent probes have
been conjugated to high molecular weight polysaccharides for
assessing tumor targeting and cellular uptake of corresponding
drug-conjugated polysaccharides. These approaches each have limited
capabilities including: overly high specificity for populations of
cells, cell surfaces and extracellular components; lack of
targeting to certain components of cell surfaces and extracellular
components; and physicochemical properties that preclude their
binding ability to target certain tumor cell surfaces.
[0002] It is an object of the present invention to provide
fluorescently labeled conjugates as probes for tumor targeting and
cellular uptake of low molecular weight oligosaccharide fluorophore
conjugates.
[0003] It is another object of the present invention to provide
fluorescently labeled conjugates for immunofluorescent staining,
confocal microscopic imaging and flow cytometry/fluorescence
activated cell sorting (FACS).
[0004] It is a further object of the present invention to provide
fluorescently labeled monosaccharide and low molecular weight
oligosaccharide conjugates having improved physicochemical
properties that permit their binding ability to certain tumor cell
surfaces and extracellular components.
BRIEF SUMMARY OF THE INVENTION
[0005] The present invention relates to fluorescent probes
comprising targetable conjugates of a fluorescent compound and
glycosides having the formula
##STR00001##
where R is an alkylene or heteroalkylene, Y=0 or 1, R.sub.1,
R.sub.2 and R.sub.3 are independently hydrogen, alkyl, aryl,
aralkyl and cycloalkyl, R.sub.4 is independently hydrogen, a
monomeric glycoside, a dimeric glycoside and alkyl, X=0 or 1, Z is
oxygen or a linker, n is from 1 to about 4 and R.sub.5 is hydrogen
or a fluorophore with the proviso that said compound contains at
least one fluorophore.
DETAILED DESCRIPTION OF THE INVENTION
[0006] In accordance with the present invention, the fluorescent
conjugates comprise a fluorophore attached to monosaccharides or
oligosaccharides having a molecular weight less than about 2,000
Daltons. In accordance with a preferred embodiment of the present
invention, the fluorescent conjugates comprise a fluorophore
covalently attached to water-soluble monosaccharides or to
oligosaccharides having from 2 to about 8 glycoside residues. In a
more preferred embodiment of the present invention, the fluorescent
conjugates comprise a fluorophore covalently attached to
water-soluble monosaccharides or to oligosaccharides having from 2
to about 6 glycoside residues.
[0007] Unless otherwise specifically identified or claimed for
preferred embodiments, the following general definitions are used
in accordance with the present invention.
[0008] In accordance with the present invention, the term
fluorophore refers to a fluorescent chemical moiety which absorbs
light at a given wavelength and emits light at a higher
wavelength.
[0009] In accordance with the present invention, the term
targetable refers to the recognition of a target by the conjugates.
Targets include cell surfaces and extracellular components
including, but not limited to tumor-specific antigens, negatively
charged sialic acid residues on tumor cells, leukocytes and
components of the extracellular matrix, and sialic acid on mucosal
surfaces.
[0010] In accordance with the present invention, the term alkyl
refers to a branched or straight chain acyclic alkyl group
comprising one to about ten carbon atoms. In accordance with the
present invention, the term lower alkyl refers to a branched or
straight chain acyclic alkyl group comprising one to about six
carbon atoms.
[0011] In accordance with the present invention, cycloalkyl refers
to a saturated or unsaturated cyclic hydrocarbon comprising from
about 3 to about 8 carbon atoms. Cycloalkyl groups can be
unsubstituted or substituted with one, two or three substituents
independently selected from alkyl, alkoxy, amino, alkylamino,
dialkylamino, arylamino, diarylamino, alkylarylamino, aryl, amidyl,
ester, hydroxy, halo, carboxyl, alkylcarboxylic acid,
alkylcarboxylic ester, carboxamido, alkylcarboxamido, oxo and
nitro. In accordance with the present invention, cycloalkyl groups
include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cyclohexenyl, cycloheptyl and the like.
[0012] In accordance with the present invention, the term aryl
refers to an unsubstituted or substituted monocyclic, bicyclic,
carbocyclic or heterocyclic ring system comprising one or two
aromatic rings. In accordance with the present invention, aryl
groups include phenyl, pyridyl, napthyl, quinoyl,
tetrahydronaphthyl, furanyl, indanyl, indenyl, indoyl, and the
like.
[0013] In accordance with the present invention, the term arylalkyl
refers to an aryl radical, attached to an alkyl radical in
accordance with the present invention,
[0014] In accordance with the present invention tumor targeting and
cellular uptake of the water-soluble low molecular weight
oligosaccharide fluorescent conjugates can easily be determined by
a number of conventional techniques described in the scientific
literature including immunofluorescent staining, fluorescence
microscopy, confocal microscopic imaging and flow
cytometry/fluorescence activated cell sorting (FACS). Many of the
classical fluorescent probes that have been successfully utilized
in confocal microscopic imaging and flow cytometry/fluorescence
activated cell sorting (FACS) include fluorescein isothiocyanate
(FITC), rhodamine analogs, and Texas Red.
[0015] Fluorescein is a xanthene dye that has a high quantum yield,
an absorption maximum at 495 nanometers and coincides quite well
with the 488 nanometer (blue) spectral line produced by argon-ion
and krypton-argon lasers, as well as the 436 of the mercury lamps
and the 467 principal lines of the xenon arc-discharge lamps.
Rhodamine dyes include, Rhodamine 123, Rhodamine B
(tetraethylrhodamine), tetramethylrhodamine isothiocyanate (TRITC),
NHS-Rhodamine, Rhodamine 116, Rhodamine 110, and Rhodamine 6G.
TRITC is the base tetramethylrhodamine molecule functionalized with
an isothiocyanate reactive group (--N.dbd.C.dbd.S) at one of two
hydrogen atoms on the bottom ring of the structure. NHS-Rhodamine
is activated with the N-hydroxy-succinimidyl-ester (NHS ester)
functional group. NHS-ester derivative has high specificity toward
primary amines, and results in a more stable linkage following
labeling. Texas Red is a long-wavelength derivative of rhodamine
that is modified with sulfonyl chloride for reaction to primary
amines.
[0016] BODIPY fluorophores have spectral characteristics different
than those of fluorescein, tetramethylrhodamine, Texas Red and
longer-wavelength dyes. These fluorophores are useful as conjugates
of proteins, nucleotides, oligonucleotides and dextrans, as well as
to prepare fluorescent enzyme substrates, fatty acids,
phospholipids, lipopolysaccharides, receptor ligands and
polystyrene microspheres. BODIPY is an unsubstituted dipyrromethene
referred to as 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene.
BODIPY-FL is comprised of dipyrromethene complexed with a
disubstituted boron atom, typically a BF2 unit, and is called
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-/s/-indacene.
[0017] The use of the fluorophores in fluorescence-activated cell
sorting (FACS) and immunohistochemical analysis is well-known and
is clearly documented in the scientific and patent literature as
evidenced by the following United States Patents which are hereby
incorporated by reference: U.S. Pat. No. 7,505,618; U.S. Pat. No.
4,395,397; U.S. Pat. No. 4,629,687; U.S. Pat. No. 5,035,693; U.S.
Pat. No. 4,284,897; U.S. Pat. No. 5,381,224; U.S. Pat. No. 5,646,41
1; U.S. Pat. No. 5,672,880; U.S. Pat. No. 5,7 19,391; U.S. Pat. No.
4,532,402; U.S. Pat. No. 5,690,846; U.S. Pat. No. 5,296,963; U.S.
Pat. No. 5,932,872 and U.S. Pat. No. 7,462,696.
[0018] The preparation and use of the fluorophore probes in
fluorescence-activated cell sorting (FACS), fluorescent
immunohistochemical analysis and fluorophore conjugates targeted to
tumor cells is well-known and is documented in the scientific
literature including, Luo et al. Biomacromolecules 1:208-218
(2000); Pouyani et al. Bioconjugate Chemistry 5:370-372 (1994);
Prestwich et al. Journal of Controlled Release 53 :93-103 (1998);
Luo et al. Bioconjugate Chemistry 10: 755-753 (1999); Wortzel et
al. EP 1991587 (WO 2007/102149) and Pouyani et al. Bioconjugate
Chemistry 5:339-347 (1994).
[0019] In accordance with the present invention, disclosed are
targetable fluorophore conjugates of having the formula
##STR00002## [0020] where R is an alkylene or heteroalkylene, Y=0
or 1, R.sub.1, R.sub.2 and R.sub.3 are independently hydrogen,
alkyl, aryl, aralkyl and cycloalkyl, R.sub.4 is independently
hydrogen, a monomeric glycoside, a dimeric glycoside or alkyl, X=0
or 1, Z is oxygen or a linker, n is from 1 to about 4 and R.sub.5
is hydrogen or a fluorophore with the proviso that said compound
contains at least one fluorophore.
[0021] In accordance with the present invention, R is an alkylene
containing only carbon atoms or a heteroalkylene containing carbon,
nitrogen and oxygen atoms. Examples of heteroalkylenes include
--NHC(O)CH.sub.2-- and --NHC(O)CH.sub.2CH.sub.2--. In accordance
with the present invention, R is an alkylene having from 1 to about
6 carbon atoms. In a preferred embodiment of the present invention,
R is a lower alkylene having from 1 to about 3 carbon atoms. In
accordance with a more preferred embodiment of the present
invention, R is --CH.sub.2 or --CH.sub.2CH.sub.2--. In accordance
with the present invention, Y is either 0 or 1. In accordance with
a preferred embodiment of the present invention, Y is 0.
[0022] In accordance with the present invention, R.sub.1, R.sub.2
and R.sub.3 are independently hydrogen, alkyl, aryl and cycloalkyl.
Preferred alkyls are those having from 1 to about 6 carbon atoms
including but not limited to methyl, ethyl, propyl, butyl,
isobutyl, pentyl and hexyl. More preferably the alkyls contain from
1 to about 3 carbon atoms. In accordance with the present
invention, R.sub.1, R.sub.2 and R.sub.3 are each methyl or ethyl.
Preferred aryls are those including but not limited to phenyl and
pyridinyl, while a preferred aralkyl is benzyl.
[0023] Preferred cycloalkyls are those having from about 3 to about
6 carbon atoms including but not limited to cyclopropyl,
cyclobutyl, cyclopentyl, and cyclohexyl.
[0024] In accordance with the present invention, R.sub.4 is
independently hydrogen, a monomeric glycoside, a dimeric glycoside
or alkyl. In accordance with one aspect of the present invention
R.sub.4 is an alkyl having from 1 to about 6 carbon atoms. In a
preferred embodiment of the present invention, R.sub.4 is
independently hydrogen, methyl, ethyl or propyl. In accordance with
the present invention, illustrative dimeric glycosides include, but
are not limited to, compounds having the formula
##STR00003##
[0025] In accordance with this embodiment of the present invention,
the oligosaccharides include multiple glycosides such that n is an
integer from about 1 to about 4. In a preferred embodiment of the
present invention, the conjugates include an oligosaccharide where
n is an integer from 1 to about 3.
[0026] In accordance with the present invention, the linker can be
any suitable chemical group that permits conjugation of the
fluorescent compound with the carboxyl or hydroxyl group of the
saccharides including, but not limited to, hydrazides, hydrazones,
succinates, adipates, suberates, and adipic dihydrazide.
[0027] Illustrative compounds in accordance with another aspect of
the present invention include, inter alia,
fluorophore-oligosaccharide conjugates having the formula:
##STR00004##
where n=1 to about 4; fluorophore-oligosaccharide conjugates having
the formula
##STR00005##
where n=1 or 2, and fluorophore-oligosaccharide conjugates having
the formula
##STR00006##
[0028] Illustrative compounds in accordance with another aspect of
the present invention include, inter alia,
fluorescein-oligosaccharide conjugates having the formula:
##STR00007##
[0029] Illustrative compounds in accordance with another aspect of
the present invention include, inter alia, Texas
Red-oligosaccharide conjugates having the formula:
##STR00008##
[0030] In another aspect of the present invention, disclosed are
monosaccharide drug conjugates having the formula
##STR00009##
where R is an alkylene or heteroalkylene, Y=0 or 1, R.sub.1,
R.sub.2 and R.sub.3 are independently hydrogen, alkyl, aryl,
aralkyl and cycloalkyl, X=0 or 1, Z is oxygen or a linker and
R.sub.5 is a fluorophore. In a preferred embodiment of the present
invention, the fluorophore is conjugated to 2-amino-D-glucuronic
acid (CAS No. 50767-834).
[0031] The present invention has been described in detail using
specific examples to illustrate the preferred embodiments of the
invention; however, it will be obvious to those skilled in the art
that various modifications thereto can be made without departing
from the spirit and scope thereof.
* * * * *